Method for microbial production of difructose anhydride III , micro-organism used therefor and enzyme with inulase II activity and dna sequences coding therefor

ABSTRACT

The invention relates to a method for the production of difructose anhydride III by enzymatic decomposition of inulase using an enzyme with inulase II activity. Said enzyme can be obtained from a microorganism of the Arthrobacter sp. Bu0141 species. The invention also relates to DNA sequences derived therefrom comprising a region coding for said enzyme in addition to plasmides and micro-organisms containing said DNA sequences.

[0001] The present invention relates to a process for the microbialproduction of difructose anhydride III, a microorganism which issuitable for this process and has the ability to express an enzyme withinulase II activity, an enzyme with inulase II activity and DNAsequences with a region coding for this enzyme.

[0002] Difructose anhydride III is a disaccharide which contains twofructose units linked to one another via 1-2′ and 2-3′ bonds.

[0003] Difructose anhydride III (DFA III) can be obtained by microbialdecomposition of inulin by the enzyme inulase II, a transferase.

[0004] It is known that the enzyme inulase II can be produced by somemicroorganisms. These include various species of the genus Arthrobacter,such as, for example, Arthrobacter ureafaciens 7116, Arthrobacterglobiformis C 11-1, Arthrobacter aurescens IFO 12136 and Arthrobacterilicis MCI-2297, and of the genus Pseudomonas, such as Pseudomonasfluorescens no. 949.

[0005] A process for the microbial decomposition of inulin to DFA III bymeans of Arthrobacter ilicis is described in EP 0 332 108 B1, the enzymewith inulase II activity obtained from this microorganism showing amaximum activity at 60° C. and being stable for a short time up to atemperature of 70° C. However, there is no information on the period oftime and the residual activity.

[0006] There was, however, a demand for further improved processes, inparticular for processes which can be carried out with easily obtainableand accessible (recombinant) microorganisms, and for enzymes which stillhave high residual activities, preferably of up to 100%, over a longperiod of time, for example several hours, even at elevated temperature.

[0007] The object of the present invention was therefore to provide aprocess for the microbial production of difructose anhydride III whichcan be carried out with easily obtainable accessible microorganismswhich can obtain DFA III from inulin with a high efficiency.

[0008] It was also an object of the invention to provide an enzyme withinulase II activity which has a high heat stability over a long periodof time.

[0009] To achieve the object, the present invention provides, inparticular, DNA sequences which code for an enzyme with inulase IIactivity, and microorganisms which contain and can express this gene andwhich can advantageously be used for a process for the microbialproduction of DFA III.

[0010] The present invention therefore relates to DNA sequences whichcode for an enzyme with inulase II activity, characterized by that afterintroduction of these DNA sequences into a microorganism, there occursexpression of the enzyme with inulase II activity which effects thedecomposition of inulin to DFA III.

[0011] The invention relates in particular to DNA sequences which codefor an enzyme with inulase II activity, comprising

[0012] a nucleotide sequence according to sequence no. 1, sequence no. 2or sequence no. 3 as shown in the Figures;

[0013] a nucleotide sequence which comprises the region according to oneof sequences no. 1 to 3 which codes for an enzyme with inulase IIactivity, and

[0014] a nucleotide sequence which codes for an enzyme which comprisesthe amino acid sequences shown for sequences no. 1 to 3.

[0015] A reproduction of the sequences is to be found in the sequencelisting section of the description:

[0016] DNA sequence no. 1 with the amino acid sequence derivedtherefrom.

[0017] DNA sequence no. 2 with the amino acid sequence derivedtherefrom, and

[0018] DNA sequence no. 3.

[0019] The invention furthermore relates to a microorganism of the genusArthrobacter which contains one of the abovementioned DNA sequences, andto plasmids and recombinant microorganisms which contain one of theabovementioned DNA sequences.

[0020] The invention furthermore relates to a process for the microbialor enzymatic production of difructose anhydride III which is carried outusing one of the abovementioned DNA sequences or a plasmid or amicroorganism which contains one of the abovementioned DNA sequences.

[0021] The Figures show

[0022]FIG. 1 the enzymatic synthesis of DFA II andfructo-oligosaccharides from inulin;

[0023]FIG. 2 the gene map of the Bam H1 fragments MSiftBH2 and MSiftBH1from Arthrobacter Bu0141;

[0024]FIG. 3 DNA sequence no. 1 and the amino acid sequence derivedtherefrom of the expression matrix MSiftPH with the region which codesfor active inulase II;

[0025]FIG. 4 the gene map of the plasmid pMSiftPH and modified DNAsequences derived therefrom which code for inulase II;

[0026]FIG. 5 the gene map of the plasmid pMSiftOptWT;

[0027]FIG. 6 DNA sequence no. 2 of the expression matrix MSiftOptWT andthe amino acid sequence derived therefrom; and

[0028]FIG. 7 DNA sequence no. 3 of the plasmid pMSiftOptR.

[0029] The continuations of FIGS. 3 and 6 and FIG. 7 furthermore showthe coding strand in the 5′-3′ direction (from left to right) in aseparate diagram.

[0030] The present invention also includes DNA sequences whichrepresent, for example, a fragment, derivative or allelic variant of theDNA sequences described above which code for an enzyme with inulase IIactivity. The term derivative in this connection means that thesequences differ from the DNA sequences described above at one or morepositions but have a high degree of identity to these sequences. A highdegree of identity here means a sequence of identity of more than 72.3%,including the region which codes for the signal sequence, and/or morethan 74.3% for the region which codes for the mature sub-unit,preferably above 80% and particularly preferably above 90% and inparticular at least 95% for the sequence including the signal sequenceand/or for the sequence of the mature sub-unit.

[0031] The present invention furthermore also includes DNA sequences,the complementary strand of which hybridizes with one of theabovementioned DNA sequences according to the invention and which codefor an enzyme with inulase II activity.

[0032] In the context of the present invention, the term “hybridization”means a hybridization under conventional hybridization conditions. Thisis preferably understood as hybridization under stringent conditions.

[0033] The invention includes in particular DNA sequences which have theregion according to one of sequences no. 1 to 3 which codes for themature sub-unit, or a modification thereof as described above.

[0034] The invention correspondingly also includes enzymes with inulaseII activity which can be obtained by expression of a DNA sequenceaccording to the invention, and modifications of such enzymes with anidentity of more than 74.9%, including the signal peptide, and/or morethan 77.8% for the mature sub-unit.

[0035] The DNA sequence shown in sequence no. 1 is a genomic sequencewhich comprises a coding region for an enzyme with inulase II activityfrom a microorganism Arthrobacter sp. Bu0141.

[0036] With the aid of these sequences, it is now possible for theexpert to isolate homologous sequences from other Arthrobacter speciesor strains. This can be carried out, for example, with the aid ofconventional methods, such as screening of gene libraries with suitablehybridization probes.

[0037] The DNA sequences according to the invention code for an enzymewith inulase II activity.

[0038] The microorganism Arthrobacter sp. Bu0141, the abovementionedplasmids and recombinant E. coli with plasmids pMSiftOptWT andpMSiftOptR have been deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH [German Collection ofMicroorganisms and Cell Cultures GmbH] under the following numbers andare also subject matter of the invention: Plasmid pMSiftPH DSM 13460Plasmid pMSiftOptR DSM 13461 Plasmid pMSiftOptWT DSM 13462 E. colipMSiftOptWT DSM 13463 Arthrobacter sp.Bu0141 DSM 13464 E. colipMSiftOptR DSM 13645.

[0039] This Arthrobacter strain, called Bu0141 in the following, wasisolated from a soil sample and has not been able to be assigned to anyof the species described to date. The properties of the strain Bu0141are described in more detail below.

[0040] The microorganism forms coryneform rods, is Gram-positive andstrictly aerobic and forms no acid or gas from glucose. Mobility +Spores − Catalase + meso-Diaminopimelic no acid in the cell wall:Peptidoglycan type A3α, L-Lys L-Ala₂₋₃

[0041] The sequencing of the region with the highest variability (16SrDNA sequence) gave as the highest value 97.8% agreement withArthrobacter globiformis. It can be concluded from the more than 2%differences in the 16S rDNA sequences that the microorganism is arepresentative of a species which is indeed closely related to A.globiformis but has not yet been described, and furthermore is notpathogenic.

[0042] It has been found that this strain can produce an enzyme withinulase II activity which is stable at elevated temperature over a longperiod of time. It has thus been found that the enzyme is stable at 60°C. for 140 hours with 100% residual activity.

[0043] A DNA sequence (sequence no. 1) which comprises the region whichcodes for the enzyme with inulase II activity was isolated from thisArthrobacter sp. Bu0141.

[0044] For the isolation, the ift gene (codes for inulase) was clonedfrom Arthrobacter sp. Bu0141 in λ phages, sub-cloned in E. coli andisolated in its complete length on two Bam H1 fragments. The gene map ofthese fragments, which have been called MSiftBH2 and MSiftBH1, is shownin FIG. 2.

[0045] The fragment MSiftBH2 has a length of approximately 3.2 kbp, onepart coding for the N-terminal half of the ift gene. The fragmentMSiftBH1 has a length of approx. 2.8 kbp, one part coding for theC-terminal half of the ift gene. The two Bam HI fragments were isolatedfrom the complete genomic DNA of Arthrobacter sp. Bu0141.

[0046] The singular restriction sites Pst I and Hind III, which servefor construction of an expression matrix as described below, areindicated in the gene map shown in FIG. 2. The putative ribosome bindingsite ift-RBS and the start and stop codon (ift-start and ift-stop) whichdemarcate the coding region are also marked in the gene map.

[0047]FIG. 3 shows DNA sequence no. 1 and above this the amino acidsequence derived therefrom of the Pst I/Hind III fragment identified inthe gene map in FIG. 2 with the ift gene and its surroundings fromArthrobacter sp. Bu0141. This fragment is called expression matrixMSiftPH in the following.

[0048] Expression matrix MSiftPH contains 1,884 nucleotides.

[0049] The enzyme with inulase II activity is coded by 1,350 nucleotidesand comprises 450 amino acids. The first 40 amino acids serve as thesignal peptide and ensure transport of the expressed enzyme from thecell. This signal peptide is cut off during or after transport of theenzyme from the cell in Arthrobacter sp. Bu0141. The mature sub-unit ofthe enzyme with inulase II activity itself comprises 410 amino acids.

[0050] The putative ribosome binding site ift-RBS with the start codonGTG, the stop codon (*) and the presumed cleavage site between thesignal peptide and the coding region of the mature sub-unit (▾) arefurthermore identified in the sequence shown in FIG. 3.

[0051] Starting from the fragment MSiftPH, foreign expression systemshave now been developed according to the invention, which can beintroduced into a host organism and can effect expression of an enzymewith inulase II activity in this host organism.

[0052] For this, the above DNA fragment MSiftPH or parts thereof whichcontain the coding region for the enzyme with inulase II activity werelinked to the elements which are suitable for the particular hostorganism and control transcription, such as promoter and stop codon, itbeing possible for the DNA sequence to be modified before or after thelinking if required.

[0053] For example, all or part of the signal sequence was removed fromthe coding DNA, since as a rule this can be neither recognized by a hostorganism for export from the cell nor cleaved posttranslationally.

[0054] It has been found here that by shortening or complete removal ofthe signal sequence, a significant increase in the enzyme activity canbe effected. The results of these deletion experiments are described inthe following.

[0055] With the aid of DNA sequence 1 shown under FIG. 3 or partsthereof which contain the coding region for the enzyme with inulase IIactivity, it is possible to modify microorganisms to the extent thatthey express active inulase II.

[0056] For preparation for introduction of foreign genes intomicroorganisms, a large number of cloning vectors which contain theelements for control of expression required for a particularmicroorganism are available. The desired sequence can be introduced intothe vector at an appropriate restriction cleavage site. Any plasmid DNAsequence can be cloned into the same vector or into other plasmids bythis procedure. The techniques, vectors and appropriate control elementsare known per se and can easily be chosen and/or adapted for theparticular host organism to be transformed.

[0057] The production of recombinant host organisms according to theinvention which contain a DNA sequence according to the invention andhave the ability to express active inulase II is described in thefollowing by the example of transformation of E. coli, expressionconstructs which contain DNA sequence I shown in FIG. 3 or parts thereofwith the region which codes for inulase II being introduced into themicroorganism. pUC 18 and pUC 19 were used as vectors for the followingexample. The DNA fragments MSiftPH according to FIG. 3 and modificationsthereof which contained the region which codes for active inulase IIwere introduced into these vectors and the corresponding enzyme activityof E. coli transformed therewith was investigated.

[0058]FIG. 4 shows the gene map of the inulase expression constructpMSiftPH obtained, the expression matrix MSiftPH, which is shown in FIG.3, having been integrated into the commercially obtainable vector pUC18. The expression construct pMSiftPH was transformed into E. coli. Thequality of the expression construct was checked in the inulase activitytest described in the following. Transformants with the expressionconstruct pMSiftPH showed a significant inulase activity of about 3,600U/l.

[0059] Deletion experiments were undertaken in order to investigate theinfluence of the signal peptide on the enzyme activity. It was shown inthese that by shortening the signal peptide at the DNA level it waspossible to increase the inulase activity of the expression constructpMSiftPH 20-fold. Thus, an expression product (enzyme) with only 456amino acids compared with 477 amino acids for the expression product(enzyme) of MSiftPH shows an activity of about 14,000 U/l (FIG. 4c) andthe corresponding expression product (enzyme) with 431 amino acids showsan activity of about 70,000 U/l (FIG. 4d).

[0060] A DNA sequence (sequence no. 2) which codes for active inulase IIand in which the DNA sequence which codes for the signal peptide hasbeen completely removed is shown in FIG. 6. Nucleotide sequence no. 2shown in FIG. 6 is called expression matrix MSiftOptWT in the following.In this sequence the region which codes for the mature inulase sub-unitwithout the signal peptide starts at nucleotide position 25.

[0061] The expression matrix MSiftOptWT was tested for efficiency inseveral vectors. It was found here that it was possible to achieve anincrease in inulase activity by a factor of 2 to 3 solely by cloning thesame expression matrix from plasmid pUC 18 to pUC 19.

[0062] The expression construct pMSiftOptWT was prepared from theexpression matrix MSiftOptWT and plasmid pUC 19 by integrating theMSiftOptWT fragment, optimized to a nucleotide, directly into thereading frame in pUC 19 which starts at the Lac RBS via thesynthetically produced Hind III or Eco RI cleavage sites.

[0063] The gene map of the pMSiftOptWT expression constructs obtained,of the combination of expression matrix MSiftOptWT and the plasmid pUC19, is shown in FIG. 5. The inulase activity of an E. coli transformedwith the vector pMSiftOptWT was greater than 320,000 U/l.

[0064] A fusion protein with the amino acid sequence shown in FIG. 6,which comprises 418 amino acids, was obtained as the expression product,the enzyme with inulase activity starting at amino acid position 9 ADGQQ. . .

[0065]FIG. 7 shows DNA sequence no. 3 of a plasmid pMSiftOptR whichdiffers from DNA sequence no. 2 of plasmid pMSiftOptWT in one nucleotideat position 661 in that nucleotide G of sequence no. 2 has been replacedby nucleotide A in sequence no. 3, as a result of which R (Arg) isincorporated instead of G (Gly) at position 221 in the correspondingamino acid sequences. This minor modification causes an increase inactivity to 435,000 U/l, that is to say an increase by a factor of 1.35.

[0066] The procedure for the experiments and the results of an inulaseactivity test carried out with the expression products (enzymes)obtained are described in the following.

[0067] Procedure for the inulase activity test

[0068] The test described in the following served merely for a rapidcomparative analysis, and it is to be expected that the values for theenzyme activity will be several times higher on a preparative scaleunder optimized conditions, such as larger number of cells, moreeffective cell disruption etc.

[0069] The strains were cultured by inoculating 5 ml Luria-Bertanimedium, to which 60 μg/ml ampicillin had been added, with a singlecolony of E. coli, which had been transformed with the particularexpression construct (plasmid), and shaking the culture for 16 hours at37° C. and 170 rpm.

[0070] The host organism used was an E. coli strain from Stratagene® {E.coli XL1-blue MRF′ Kan: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI^(q)ZΔM15 Tn5 (Kan′)]^(c)}.

[0071] The inulase II expression was intracellular; the enzyme reactionand DFA III formation took place in the cell-free extract afterdisruption of the cells.

[0072] For the activity test, in each case 0.5 ml of the freshexpression culture described above was used, 0.5 ml of the culture beingpelleted, the supernatant being discarded and the cells beingresuspended in 5 ml of cooled 0.9% NaCl solution.

[0073] The cell disruption was carried out by means of ultrasonification(KE 76, cont. 50%, 60 sec; Bandelin, Sonopulus HD 200).

[0074] 1 ml of the disrupted cells was removed and pelleted in a benchcentrifuge for 10 min (20,000×g). 100 μl of the enzyme-containingsupernatant were transferred to 1,000 μl of a 10% inulin solution (pH5.5) and incubated for 30 min at 50° C.

[0075] The enzyme reaction was stopped by heating to 100° C. for 10 minand the solution was centrifuged in a bench centrifuge (10 min,20,000×g).

[0076] 100 μl of the supernatant, which contained the product DFA III,were transferred into 1,000 μl HPLC eluent and the product DFA III wasmeasured by means of HPLC.

[0077] A value of product formation of approximately 2.6 g/l resultedhere for the clone of the expression construct pMSiftOptWT, whichcorresponded to an enzyme activity of approx. 323,000 U/l (one unit=1μmol/min).

[0078] For the clone of the expression construct pMSiftOptR, whichdiffers from expression construct pMSiftOptWT in a single nucleotide atposition 661 by replacement of G (sequence no. 2) by A (sequence no. 3),a value of 3.5 g/l DFA III was found for the product formation, whichcorresponded to an enzyme activity of approx. 435,000 U/l. Compared withthe expression construct pMSiftOptWT, an increase of 1.35-fold is thusobserved.

[0079] The corresponding enzyme activities for clones of the expressionconstruct with plasmid pUC18 and of expression matrix MSiftPH with acomplete signal sequence corresponding to an expression product with 477amino acids, with a shortened signal sequence corresponding to anexpression product with 456 amino acids or corresponding to anexpression product with 431 amino acids, and with expression matrixMSiftOptWT without a signal sequence were approx. 3,500, approx. 14,000,approx. 70,000 and approx. 120,000 U/l.

[0080] On the basis of their high heat stability, the enzymes withinulase II activity obtainable from Arthrobacter sp. Bu0141 and the DNAsequences isolated or derived therefrom which code for an enzyme withinulase II activity are outstandingly suitable for a process for theenzymatic decomposition of inulin for the production of difructoseanhydride III. Due to the possibility described of transforming andexpressing these DNA sequences in generally available host organisms,tailor-made recombinant microorganisms with a high enzyme activity canbe obtained, the enzymes expressed having a high heat stability andtherefore being able to decompose inulin to difructose anhydride IIIefficiently.

1 3 1 1884 DNA Arthrobacter sp. CDS (35)..(1384) mat_peptide(155)..(1384) 1 ctgcagcagt cttatcccat acaaaggaga cccc gtg gta act ggcaag aat cta 55 Val Val Thr Gly Lys Asn Leu -40 -35 gac aaa gcg aat ccaagc cgc cgt cgg ctg atc ggc gcc gga gcc gcc 103 Asp Lys Ala Asn Pro SerArg Arg Arg Leu Ile Gly Ala Gly Ala Ala -30 -25 -20 gga acc ctg gcg gctgcc ttg acc ctc ggg acg atg cag aac gcc aat 151 Gly Thr Leu Ala Ala AlaLeu Thr Leu Gly Thr Met Gln Asn Ala Asn -15 -10 -5 gcg gcc gac ggc cagcaa ggt acc ccc ctc aat tcg ccc aac acg tac 199 Ala Ala Asp Gly Gln GlnGly Thr Pro Leu Asn Ser Pro Asn Thr Tyr -1 1 5 10 15 gac gta acc aca tggagg atc aag gca cac ccg gac gtc acc gcg cag 247 Asp Val Thr Thr Trp ArgIle Lys Ala His Pro Asp Val Thr Ala Gln 20 25 30 tcc gac att ggg gcg gtcatc aac gac atc atc gcc gac atc aag caa 295 Ser Asp Ile Gly Ala Val IleAsn Asp Ile Ile Ala Asp Ile Lys Gln 35 40 45 cgg cag acg tca ccg gac gcgcgt ccc gga gcc gcg atc att atc cca 343 Arg Gln Thr Ser Pro Asp Ala ArgPro Gly Ala Ala Ile Ile Ile Pro 50 55 60 ccg ggc gac tac gac ctg cac acccag gtc gtc gtc gac ata agt tac 391 Pro Gly Asp Tyr Asp Leu His Thr GlnVal Val Val Asp Ile Ser Tyr 65 70 75 ctg aca atc gcg ggc ttc ggg cat ggcttc ttc tcc cga agc atc ctc 439 Leu Thr Ile Ala Gly Phe Gly His Gly PhePhe Ser Arg Ser Ile Leu 80 85 90 95 gac aac tcg aac ccg acc gga tgg cagaac ctc caa ccc gga gca agc 487 Asp Asn Ser Asn Pro Thr Gly Trp Gln AsnLeu Gln Pro Gly Ala Ser 100 105 110 cac atc cgc gtc ctg acc tct ccg agcgcg ccc cag gca ttc ctc gtc 535 His Ile Arg Val Leu Thr Ser Pro Ser AlaPro Gln Ala Phe Leu Val 115 120 125 cgc cgg aca ggg gat ccc cgt ctt tcagga atc gtg ttc cgg gac ttc 583 Arg Arg Thr Gly Asp Pro Arg Leu Ser GlyIle Val Phe Arg Asp Phe 130 135 140 tgc ctc gac gga gtc ggc ttc acc cccgac aag aac agc tac cac aac 631 Cys Leu Asp Gly Val Gly Phe Thr Pro AspLys Asn Ser Tyr His Asn 145 150 155 ggc aag acc gga atc gaa gtc gcc tccgac aac gac tcc ttc cac atc 679 Gly Lys Thr Gly Ile Glu Val Ala Ser AspAsn Asp Ser Phe His Ile 160 165 170 175 acc ggc atg gga ttc gtc tac ctcgaa cat gcc ctg atc gtg cgc ggc 727 Thr Gly Met Gly Phe Val Tyr Leu GluHis Ala Leu Ile Val Arg Gly 180 185 190 gcc gac gcg ctc cgc gtc aac gacaac atg atc gcc gaa tgc ggc aac 775 Ala Asp Ala Leu Arg Val Asn Asp AsnMet Ile Ala Glu Cys Gly Asn 195 200 205 tgc gtc gag ctc acc ggg gcc gggcag gcc aca att gtc agc ggc aat 823 Cys Val Glu Leu Thr Gly Ala Gly GlnAla Thr Ile Val Ser Gly Asn 210 215 220 cac atg ggc gcc ggc cct gac ggggta acc ctc ctg gcc gag aac cac 871 His Met Gly Ala Gly Pro Asp Gly ValThr Leu Leu Ala Glu Asn His 225 230 235 gag ggc ctc ctc gtc acc ggc aacaac ctc ttc cca cgc ggc cgc agc 919 Glu Gly Leu Leu Val Thr Gly Asn AsnLeu Phe Pro Arg Gly Arg Ser 240 245 250 255 ctc atc gaa ctc acc ggc tgcaac cgg tcc tca gtc tcc tcg aac agg 967 Leu Ile Glu Leu Thr Gly Cys AsnArg Ser Ser Val Ser Ser Asn Arg 260 265 270 ctc cag ggc ttt tac ccg ggcatg ctc cgc ctg ctg aac ggc tgc aag 1015 Leu Gln Gly Phe Tyr Pro Gly MetLeu Arg Leu Leu Asn Gly Cys Lys 275 280 285 gag aac ctc atc acg gcc aaccac atc cgc cgg acc aac gag ggg tac 1063 Glu Asn Leu Ile Thr Ala Asn HisIle Arg Arg Thr Asn Glu Gly Tyr 290 295 300 ccg ccg ttc atc ggc cgc ggcaac ggc ctc gac gac ctc tac ggc gtc 1111 Pro Pro Phe Ile Gly Arg Gly AsnGly Leu Asp Asp Leu Tyr Gly Val 305 310 315 gtc cac atc gcg gga gac aacaac ctc atc tcg gac aac ctc ttc gcc 1159 Val His Ile Ala Gly Asp Asn AsnLeu Ile Ser Asp Asn Leu Phe Ala 320 325 330 335 tac aac gtc ccg ccc ggcaac atc gcc ccc gcc ggc gcc cag ccg acc 1207 Tyr Asn Val Pro Pro Gly AsnIle Ala Pro Ala Gly Ala Gln Pro Thr 340 345 350 cag atc ctg atc gcc ggcgga gac gcc aac gtg gtg gcg ctc aac cac 1255 Gln Ile Leu Ile Ala Gly GlyAsp Ala Asn Val Val Ala Leu Asn His 355 360 365 gtg gtc agc gac gtc gcttcc cag cac gtc gtt ctg gac gca tcc acc 1303 Val Val Ser Asp Val Ala SerGln His Val Val Leu Asp Ala Ser Thr 370 375 380 act cac tcg aaa gtg ctcgac agc ggt acc gcc tcc cag atc acc tcg 1351 Thr His Ser Lys Val Leu AspSer Gly Thr Ala Ser Gln Ile Thr Ser 385 390 395 tac agc acg gac acc gctatc cgg ccg acc ccc tgacaggcgg agagcagctt 1404 Tyr Ser Thr Asp Thr AlaIle Arg Pro Thr Pro 400 405 410 ctcggaaacc accggacgcg ccaagggcatttcttatgtt ggggcccgga ccaatcggtg 1464 atatcgcggg gagcctcagc ggtccttgagaggctccccg atcaattcgg gctgccggtt 1524 gctccagtcg tggaagtagg gagcggcgccgtggtggtgc ttgttgttgt actcctgggc 1584 aagacccagt gcaccttcga gcccggggaagacccggtct ttggtgtgat cagcgcatct 1644 gacgaggaaa ccgagccccc taaagccgtagcactgggtt acataagcgg gtcgagtcga 1704 aatgtccccc ttggtgtcgt tccgccctccgacggggccc gcttagatgg ttctatctcc 1764 ggaatcctga tctacctcag tcactggtgatttgatccat gtgacgacca cactcacccc 1824 gccgtcctcg tcccgttcgg tctcgatttcaatctcggaa gccgacgccc caataagctt 1884 2 1737 DNA Arthrobacter sp. CDS(1)..(255) mat_peptide (25)..(255) 2 atg acc atg att acg cca agc ttg gccgac ggc cag caa ggt acc ccc 48 Met Thr Met Ile Thr Pro Ser Leu Ala AspGly Gln Gln Gly Thr Pro -5 -1 1 5 ctc aat tcg ccc aac acg tac gac gtaacc aca tgg agg atc aag gca 96 Leu Asn Ser Pro Asn Thr Tyr Asp Val ThrThr Trp Arg Ile Lys Ala 10 15 20 cac ccg gac gtc acc gcg cag tcc gac attggg gcg gtc atc aac gac 144 His Pro Asp Val Thr Ala Gln Ser Asp Ile GlyAla Val Ile Asn Asp 25 30 35 40 atc atc gcc gac atc aag caa cgg cag acgtca ccg gac gcg cgt ccc 192 Ile Ile Ala Asp Ile Lys Gln Arg Gln Thr SerPro Asp Ala Arg Pro 45 50 55 gga gcc gcg atc att atc cca ccg ggc gac tacgac ctg cac acc cag 240 Gly Ala Ala Ile Ile Ile Pro Pro Gly Asp Tyr AspLeu His Thr Gln 60 65 70 gtc gtc gtc gac ata agttacctga caatcgcgggcttcgggcat ggcttcttct 295 Val Val Val Asp Ile 75 cccgaagcat cctcgacaactcgaacccga ccggatggca gaacctccaa cccggagcaa 355 gccacatccg cgtcctgacctctccgagcg cgccccaggc attcctcgtc cgccggacag 415 gggatccccg tctttcaggaatcgtgttcc gggacttctg cctcgacgga gtcggcttca 475 cccccgacaa gaacagctaccacaacggca agaccggaat cgaagtcgcc tccgacaacg 535 actccttcca catcaccggcatgggattcg tctacctcga acatgccctg atcgtgcgcg 595 gcgccgacgc gctccgcgtcaacgacaaca tgatcgccga atgcggcaac tgcgtcgagc 655 tcaccggggc cgggcaggccacaattgtca gcggcaatca catgggcgcc ggccctgacg 715 gggtaaccct cctggccgagaaccacgagg gcctcctcgt caccggcaac aacctcttcc 775 cacgcggccg cagcctcatcgaactcaccg gctgcaaccg gtcctcagtc tcctcgaaca 835 ggctccaggg cttttacccgggcatgctcc gcctgctgaa cggctgcaag gagaacctca 895 tcacggccaa ccacatccgccggaccaacg aggggtaccc gccgttcatc ggccgcggca 955 acggcctcga cgacctctacggcgtcgtcc acatcgcggg agacaacaac ctcatctcgg 1015 acaacctctt cgcctacaacgtcccgcccg gcaacatcgc ccccgccggc gcccagccga 1075 cccagatcct gatcgccggcggagacgcca acgtggtggc gctcaaccac gtggtcagcg 1135 acgtcgcttc ccagcacgtcgttctggacg catccaccac tcactcgaaa gtgctcgaca 1195 gcggtaccgc ctcccagatcacctcgtaca gcacggacac cgctatccgg ccgaccccct 1255 gacaggcgga gagcagcttctcggaaacca ccggacgcgc caagggcatt tcttatgttg 1315 gggcccggac caatcggtgatatcgcgggg agcctcagcg gtccttgaga ggctccccga 1375 tcaattcggg ctgccggttgctccagtcgt ggaagtaggg agcggcgccg tggtggtgct 1435 tgttgttgta ctcctgggcaagacccagtg caccttcgag cccggggaag acccggtctt 1495 tggtgtgatc agcgcatctgacgaggaaac cgagccccct aaagccgtag cactgggtta 1555 cataagcggg tcgagtcgaaatgtccccct tggtgtcgtt ccgccctccg acggggcccg 1615 cttagatggt tctatctccggaatcctgat ctacctcagt cactggtgat ttgatccatg 1675 tgacgaccac actcaccccgccgtcctcgt cccgttcggt ctcgatttca atctcggaat 1735 tc 1737 3 1737 DNAArthrobacter sp. 3 atgaccatga ttacgccaag cttggccgac ggccagcaaggtacccccct caattcgccc 60 aacacgtacg acgtaaccac atggaggatc aaggcacacccggacgtcac cgcgcagtcc 120 gacattgggg cggtcatcaa cgacatcatc gccgacatcaagcaacggca gacgtcaccg 180 gacgcgcgtc ccggagccgc gatcattatc ccaccgggcgactacgacct gcacacccag 240 gtcgtcgtcg acataagtta cctgacaatc gcgggcttcgggcatggctt cttctcccga 300 agcatcctcg acaactcgaa cccgaccgga tggcagaacctccaacccgg agcaagccac 360 atccgcgtcc tgacctctcc gagcgcgccc caggcattcctcgtccgccg gacaggggat 420 ccccgtcttt caggaatcgt gttccgggac ttctgcctcgacggagtcgg cttcaccccc 480 gacaagaaca gctaccacaa cggcaagacc ggaatcgaagtcgcctccga caacgactcc 540 ttccacatca ccggcatggg attcgtctac ctcgaacatgccctgatcgt gcgcggcgcc 600 gacgcgctcc gcgtcaacga caacatgatc gccgaatgcggcaactgcgt cgagctcacc 660 agggccgggc aggccacaat tgtcagcggc aatcacatgggcgccggccc tgacggggta 720 accctcctgg ccgagaacca cgagggcctc ctcgtcaccggcaacaacct cttcccacgc 780 ggccgcagcc tcatcgaact caccggctgc aaccggtcctcagtctcctc gaacaggctc 840 cagggctttt acccgggcat gctccgcctg ctgaacggctgcaaggagaa cctcatcacg 900 gccaaccaca tccgccggac caacgagggg tacccgccgttcatcggccg cggcaacggc 960 ctcgacgacc tctacggcgt cgtccacatc gcgggagacaacaacctcat ctcggacaac 1020 ctcttcgcct acaacgtccc gcccggcaac atcgcccccgccggcgccca gccgacccag 1080 atcctgatcg ccggcggaga cgccaacgtg gtggcgctcaaccacgtggt cagcgacgtc 1140 gcttcccagc acgtcgttct ggacgcatcc accactcactcgaaagtgct cgacagcggt 1200 accgcctccc agatcacctc gtacagcacg gacaccgctatccggccgac cccctgacag 1260 gcggagagca gcttctcgga aaccaccgga cgcgccaagggcatttctta tgttggggcc 1320 cggaccaatc ggtgatatcg cggggagcct cagcggtccttgagaggctc cccgatcaat 1380 tcgggctgcc ggttgctcca gtcgtggaag tagggagcggcgccgtggtg gtgcttgttg 1440 ttgtactcct gggcaagacc cagtgcacct tcgagcccggggaagacccg gtctttggtg 1500 tgatcagcgc atctgacgag gaaaccgagc cccctaaagccgtagcactg ggttacataa 1560 gcgggtcgag tcgaaatgtc ccccttggtg tcgttccgccctccgacggg gcccgcttag 1620 atggttctat ctccggaatc ctgatctacc tcagtcactggtgatttgat ccatgtgacg 1680 accacactca ccccgccgtc ctcgtcccgt tcggtctcgatttcaatctc ggaattc 1737

1. DNA sequence coding for an enzyme with inulase II activity chosenfrom a DNA sequence with a nucleotide sequence according to one ofsequences no. 1, no. 2 or no. 3, a DNA sequence which comprises theregion of sequences no. 1, no. 2 or no. 3 which codes for an enzyme withinulase II activity; a DNA sequence which codes an enzyme with inulaseII activity which comprises the amino acid sequence given for sequencesno. 1, no. 2 or no. 3; and sequences homologous to DNA sequences no. 1,2 or 3 which have an identity of more than 72.3%, including the regionwhich codes for the signal sequence, and/or more than 74.3% for theregion which codes for the mature sub-unit.
 2. Vector comprising a DNAsequence according to claim
 1. 3. Vector according to claim 2,characterized in that the vector is a plasmid pUC 18 or pUC
 19. 4.Plasmid chosen from plasmids with deposit number DSM 13460, DSM 13461and DSM
 13462. 5. Microorganism characterized in that the microorganismcontains a DNA sequence according to claim 1, a vector according to oneof claims 2 or 3 or a plasmid according to claim
 4. 6. Microorganismaccording to claim 5, characterized in that the microorganism is an E.coli with deposit number DSM 13463 or DSM
 13465. 7. Microorganism of thespecies Arthrobacter sp. with deposit number DSM
 13464. 8. Enzyme withinulase II activity, obtainable by expression of one of the DNAsequences according to claim
 1. 9. Process for the enzymaticdecomposition of inulin to difructose anhydride III, characterized inthat there is used an enzyme with inulase II activity which isobtainable via one of the DNA sequences according to claim
 1. 10.Process according to claim 9, characterized in that the DNA sequence isintroduced into a microorganism and expressed there.
 11. Processaccording to one of claims 9 or 10, characterized in that a plasmidchosen from plasmids with deposit number DSM 13460, DSM 13461 and DSM13462 or a microorganism chosen from microorganisms with deposit numberDSM 13463, DSM 13464 and DSM 13465 is used for the process.